Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.     

Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence.

Poliovirus (PV) is not often described as a monocyte- or macrophage-tropic virus; however, previous work indicated that neurovirulent PV type 1 Mahoney [PV(1)Mahoney] can productively infect primary human monocytes. To determine whether this replication has a functional role in pathogenesis, primary human mononuclear blood cells were infected with pairs of attenuated and neurovirulent strains of PV. Two neurovirulent strains of PV, PV(1)Mahoney and PV(2)MEF-1, replicated faster and to higher titers than attenuated counterparts PV(1)Sabin and PV(2)W-2, respectively, in primary human monocytes, suggesting that this replication may contribute to pathogenesis. PV(3)Leon grew weakly, while PV(3)Sabin, PV(2)Sabin, and PV(2) P712 did not replicate in these cells, perhaps because of their slow replication cycle. In U937 cells, a monocytelike cell line, PV(1)Mahoney replicated but PV(1)Sabin did not, while both grew well in HeLa cells. When molecular recombinants of PV(1)Mahoney and PV(1)Sabin were assessed, a correlation between neurovirulence and the ability to replicate in primary human mononuclear blood cells was found. Surprisingly, infectious centers assays with primary human mononuclear blood cells and U937 cells indicated that despite the lower overall viral yield, more cells are initially infected with the attenuated viruses. These results indicate that there are virulence-specific differences in the ability of PV(1)Mahoney to replicate in monocytes and suggest that there may be factors in monocytes that virulent strains of PV require.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Possible influence of measles virus infection of cynomolgus monkeys on the outcome of the neurovirulence test for oral poliovirus vaccine.

Macaque monkeys are susceptible to measles infection which triggers temporary immuno-depression similar to the well known phenomenon in humans. It is known that feral monkeys become infected with measles virus when they are exposed to humans. Since Macaca mulatta and M. fascicularis are species used to assay the neurovirulence of oral poliovirus vaccine, the immunodepression caused by measles infection of the test monkeys could significantly alter the results of the neurovirulence test. The serum titers of measles-neutralizing antibodies were studied in over 1500 monkeys used for neurovirulence tests. A high proportion of the feral monkeys had measles antibodies (51-100%); in contrast, none of 493 M. fascicularis monkeys which had been bred in a primate colony under strict isolation measures was found positive for measles antibodies. An increase in the prevalence of measles in the population of Ontario and Quebec provinces was accompanied with an increase in the proportion of measles-positive monkey and their serum antibody titers were found higher. It was observed that monkeys used in tests that had been performed during high measles prevalence presented with a poliomyelitis of more pronounced severity clinically and histologically. The analysis of 29 tests conducted on type 1 vaccines over several years showed a positive correlation (correlation coefficient = 0.5141, P less than 0.0022) between severity of poliomyelitis and the presence of measles serum antibodies in test monkeys (some animals seroconverted during the test). A similar observation, when type 3 Sabin vaccines were tested in M. fascicularis, was recently reported from another laboratory in Ontario.     

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

Determinants in the 5'' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.     

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.     

Antigenic determinants of measles virus hemagglutinin associated with neurovirulence.

The biological activity of monoclonal antibodies specific for the hemagglutinin protein of measles virus strain CAM recognizing six epitope groups according to their binding properties to measles virus strain CAM/R401 was investigated in vivo in our rat model of measles encephalitis. When injected intraperitoneally into measles virus-infected suckling rats, some monoclonal antibodies modified the disease process and prevented the necrotizing encephalopathy seen in untreated animals. The analysis of measles virus brain isolates revealed emergence of variants that resisted neutralization with the passively transferred selecting monoclonal antibody but not with other monoclonal antibodies. Monoclonal antibody escape mutants were also isolated in vitro, and their neurovirulence varied in the animal model. Sequence data from the hemagglutinin gene of measles virus localize a major antigenic surface determinant of the hemagglutinin protein between amino acid residues 368 and 396, which may be functionally important for neurovirulence. The data indicate that the interaction of antibodies with the measles virus H protein plays an important role in the selection of neurovirulent variants. These variants have biological properties different from those of the parent CAM virus.     

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.     

Structural changes associated with poliovirus inactivation in soil.

The loss of infectivity of poliovirus in moist and dried soils was a result of irreversible damage to the virus particles. The damage included (i) dissociation of viral genomes and capsids and (ii) degradation of viral ribonucleic acid (RNA) in the soil environment. Under drying conditions, capsid components could not be recovered from the soils. Further studies in sterile soils indicated that, under moist conditions, the viral RNA was probably damaged before dissociation from the capsid. However, in sterile, dried soil, RNA genomes were recovered largely intact from the soil. These results suggest that polioviruses are inactivated by different mechanisms in moist and drying soils.